Gene Set Enrichment and pathway analysis of post-mortem brain sections show enrichment of oxidative phosphorylation and NAFLD (Non-Alcoholic Fatty Liver Disease):
Three different human datasets of ALS patients pertaining to cortex, cerebellum, and muscles were identified from the GEO database (SUPPLEMENTARY-1). GSEA was carried out for the different post-mortem sections. Enrichment of cortex sections showed oxidative phosphorylation, Alzheimer's disease, Parkinson's disease, NAFLD, etc (FIGURE-1A). Enrichment analysis of the cerebellum showed enrichment of NAFLD, glutamatergic synapse, oxidative phosphorylation, Parkinson's disease, etc (FIGURE-1B). Pathway enrichment analysis for proteomic data obtained from the literature pertaining to cortex and CSF were also carried out (FIGURE-2A and 2B). The results showed enrichment of nicotinate-nicotinamide metabolism, riboflavin metabolism, alanine-aspartate-glutamate metabolism, etc. in the cortex section, and pathways related to cholesterol metabolism, vitamin absorption, etc. were enriched in CSF. Finally, commonality analysis was carried out between different brain sections and proteomics of the cortex to identify common pathways that are de-regulated. Oxidative phosphorylation and NAFLD were found to be common between different cortex sections (FIGURE-2C).
Gene set enrichment of analysis and pathway enrichment analysis of familial mice brain datasets show enrichment of oxidative phosphorylation, vitamin metabolism, signaling pathways and axonal conduction:
Mice datasets pertaining to common familial forms of the disease (SOD1, FUS, and TDP-43) were also identified from the GEO database (SUPPLEMENTARY-1). Pathway enrichment analysis and GSEA analysis of SOD1, FUS, and TDP-43 brain were carried out. The results of the individual analysis are summarized on FIGURE-3A, FIGURE-3B, and FIGURE-3C. Commonality analysis was performed to identify common pathways enriched between SOD1, FUS, and TDP-43 brain datasets. Oxidative phosphorylation and ribosome pathways were found to be common between different mice brain datasets (FIGURE-4A and 4B).
Oxidative phosphorylation was found to be common between different brain datasets:
Different brain datasets including human and mice brains showed enrichment of oxidative phosphorylation. Genes belonging to Complex-I and Complex-IV were found to be significantly enriched in the post-mortem section datasets (FIGURE-5A) and majority of the genes were down-regulated. Further, cell type analysis was carried out to identify the tissues associated with deregulated pathways. Astrocytes and glutamatergic neurons were found to be significantly affected cell types in post-mortem human datasets (FIGURE-5B and FIGURE-5C). Astrocytes are the cell types which nourish the neurons through the astrocyte-neuron Lactate shuttle 20. Dysfunction of electron transport chain in astrocytes can lead to elevated levels of lactate 21.
Pathway and cell type analysis of ALS muscle shows deregulation of oxidative phosphorylation in myofibroblast:
ALS is a neuro-muscular disease. Hence, the understanding metabolic state of muscle from ALS patients is important in understanding the disease condition. Pathway enrichment of ALS muscle biopsies showed enrichment of Parkinson's disease, Alzheimer's disease, oxidative phosphorylation, thermogenesis etc. (FIGURE-6A). Further cell type analysis showed that major metabolic pathways are deregulated in fibroblast of ALS muscle (FIGURE-6B). Based on our earlier observations, it is evident that the mitochondrial oxidative phosphorylation pathway and Complexes-I and Complex-IV in particular might be involved in ALS disease parse. Studies from literature have shown a role of deregulated mitochondrial complexes in generating reactive oxygen species associated with FUS and TDP-43. Hence, we validated our finding from ALS patients and transgenic mice model of ALS using FUS or TDP43 expressing yeast model of ALS. Saccharomyces cerevisiae has been prominently used for studying disease mechanisms, protein misfolding, amyloid aggregation, and other neurodegenerative phenotypes. Further, it has been considered as the best model system for studying amyloid diseases.
Inhibitors of mitochondrial electron transport chain and knock out of genes in Complex-III and IV confirms their role in modulating aggregation of FUS and TDP-43:
Saccharomyces cerevisiae transformed with aggressive forms of ALS (FUS, TDP-43, and their mutants) were used as a model system. Transformed cells were treated with inhibitors of mitochondrial complexes (FIGURE-7A and 7B). Complex-I and II inhibitors did not show anti-amyloid activity. However, inhibition of Complex-III and Complex-IV reduced amyloidogenesis. We also treated cells with Co-enzyme Q10, a modulator of complex-III that inhibits ROS production. Treatment with Coenzyme Q10 reduced amyloid aggregation in majority of mutants. Florescence quantification was carried out to quantitate the levels of amyloidogenesis and amyloid clearance (SUPPLEMENTARY- 2). The results obtained through quantification had a strong correlation with the imaging data. The results were further validated using knock-out studies Critical knock-outs of mitochondrial complex III and IV showed reduced amyloidogenesis in most of the mutants. Complex-IV is also known to be associated with ROS production. SOD1 and SOD2 levels are known to be directly co-related with ROS levels. Hence, SOD1 and SOD2 levels in FUS and TDP-43 transformed yeast cells were measured using the RNA sequencing technique. Our results showed that SOD1 levels were much higher in the majority of the mutants (FIGURE-7C). Higher ROS levels indicate greater mitochondrial dysfunction and thereby leading to neurodegeneration.